Use of galectin-7 to promote the re-epithelialization of wounds

ABSTRACT

Methods for the therapeutic treatment of epithelial wounds in mammals comprising administering to a mammal afflicted with an epithelial wound a therapeutically effective amount of a galectin-3 protein and/or a galectin-7 protein are provided. Pharmaceutical compositions comprising a pharmaceutically suitable carrier or diluent and as an active agent a galectin-3 protein and/or a galectin-7 protein are also provided.

RELATED APPLICATIONS

The present application claims priority to provisional application U.S.Ser. No. 60/286,903, filed Apr. 27, 2001 which is incorporated herein byreference in its entirety.

GOVERNMENT FUNDING

This invention was made with Government support under grant numberEY-07088 from the National Institutes of Health. Accordingly, thegovernment may have certain rights in this invention.

BACKGROUND OF THE INVENTION

The repair of wounds in mammalian tissue (e.g., epithelial defects,lesions, or erosions caused by disease, accidental injury, surgicalprocedure, etc.) involves an orderly, controlled cellular response.Three phases have been described in normal wound healing: acuteinflammatory phase, extracellular matrix and collagen synthesis, andremodeling (Wound Repair by Peacock, W.B. Saunders, Philadelphia, Pa.,1984). The sequence of the healing process is initiated during an acuteinflammatory phase with the deposition of provisional tissue. This isfollowed by re-epithelialization, collagen synthesis and deposition,fibroblast proliferation, and neovascularization, all of whichultimately define the remodeling phase (see, for example, Clark, J. Am.Acad Dermatol. 13:701, 1985). These events are known to be influenced bygrowth factors and cytokines secreted by inflammatory cells and byepithelial cells, endothelial cells, platelets, and fibroblastslocalized at the edges of the wound (see, for example, The Molecular andCellular Biology of Wound Repair (The Language of Science) Ed. by Clark,Plenum Press, New York, N.Y., 1996; Hunt et al., in The Surgical WoundEd. by Dineen at al., Lea & Febiger, Philadelphia, Pa., 1981; Nemeth etal., in Growth Factors and Other Aspects of Wound Healing: Biologicaland Clinical Implications Ed. by Barbul et al., A. R. Liss, New York,N.Y., 1988; and Assoian et al., Nature 309:804, 1984). Duringre-epithelialization, cells at the leading edge undergo a phenotypicconversion characterized by a dramatic reorganization of thecytoskeleton, disruption of stable intercellular adhesion, andredistribution of adhesion related molecules. The breakage of the stableintercellular contacts is a prerequisite for initiatingre-epithelialization. Following re-epithelialization, reversion to theepithelial phenotype, including the reformation of stable intercellularcontacts, must occur if the function of the epithelium is to be fullyrestored. The failure of epithelial cells to migrate over the woundsurface and failure of migrated epithelial cells to remain adherent tothe substratum are fundamental causes of debilitating clinicalconditions known as persistent epithelial defects (i.e., non healingdefects) and recurrent epithelial erosions respectively.

Disorders of wound healing constitute a serious medical problem forseveral different organ systems including the skin, gastrointestinaltract, and cornea. For example, loss of cell-cell adhesions within theepidermis produces life-threatening blistering skin diseases known aspemphigus foliaceus and pemphigus vulgaris (Cell Adhesion and HumanDisease Ed. by Marsh et al., Ciba Foundation Symposium, Vol. 189, JohnWiley & Sons, New York, N.Y., 1995). Persistent epithelial defects inthe form of delayed re-epithelialization are a characteristic of chronicskin wounds, in particular venous stasis ulcers (Falanga et al., J.Dermatol. Surg. Oncol. 19:764, 1993). Within the cornea, lack ofepithelial cell adhesion to the stroma and the basement membrane leadsto recurrent corneal erosions (Macaluso et al., in Cornea Ed. byKrachmer, Mosby, St. Louis Mo., 1997). Persistent corneal epithelialdefects occur in a wide variety of clinical situations such as ininjuries caused by radiation, corneal abrasions or lacerations, chemicalburns of the cornea such as alkali and acid burns, keratopathies,keratities and corneal dystrophies. Persistent corneal epithelialdefects carry a high risk of corneal perforation and ulceration(Macaluso et al., supra).

Despite the need for more rapid healing of wounds, to date there hasbeen only limited success in accelerating wound healing withpharmaceutical agents. In the case of corneal injuries, the use ofepidermal growth factor (Eiferman et al., Invest. Opthalmol. Vis. Sci.(Suppl.) 28:52, 1987), fibronectin (Nishida et al., J. Cell. Biol.97:1653, 1983), collagenase inhibitors (Kenyon et al., Invest.Opthalmol. Vis. Sci. 18:570, 1979), topical steroids (Lass et al., Arch.Opthalmol. 99:673, 1981), matrix metalloproteinase inhibitors (Murphy etal., Biochemistry 30:8097, 1991), ascorbates (Foster et al., Invest.Opthalmol. Vis. Sci. (Suppl.) 19:227, 1980), heparin (Aronson, Am. J.Opthalmol. 70:65, 1970), and tetracyclines (Perry et al., Ophthalmology(Suppl.) 92:77, 1985) does not always result in successful long-termmanagement. For example, topical application to a corneal injury ofepidermal growth factor (EGF) (Singh et al., Am. J. Opthalmol. 103:802,1987) or fibronectin (Tenn et al., Invest. Opthalmol. Vis. Sci. (Suppl.)26:92, 1985), enhances epithelial wound healing but does not preventrecurrent erosion and secondary breakdown of the corneal epithelialsurface.

Accordingly, there is a need in the art for additional pharmaceuticalagents and compositions that promote the healing of wounds. Inparticular, there is a need for agents, compositions and therapeuticmethods that promote the re-epithelialization of persistent epithelialdefects and prevent recurrent epithelial erosions.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for thetherapeutic treatment of epithelial injuries in mammalian tissueinvolving administering to a mammal afflicted with an epithelial injurya therapeutically effective amount of galectin-3, galectin-7, or acombination of galectin-3 and galectin-7.

In another aspect, the present invention provides pharmaceuticalcompositions that include a pharmaceutically acceptable carrier ordiluent and an amount of galectin-3 and/or galectin-7 sufficient topromote the re-epithelialization of wounds in injured mammalian tissues.

In general, it is believed that galectin-3 and/or galectin-7 will beclinically useful in promoting the healing of wounds associated with anyepithelial tissue including but not limited to the skin epithelium; thecorneal epithelium; the lining of the gastrointestinal tract; the lungepithelium; and the inner surface of kidney tubules, of blood vessels,of the uterus, of the vagina, of the urethra, or of the respiratorytract. The present invention encompasses the treatment of a variety ofwounds that include but are not limited to persistent epithelial defectsand recurrent epithelial erosions such as surgical wounds, excisionalwounds, blisters, ulcers, lesions, abrasions, erosions, lacerations,boils, cuts, sores, and burns resulting from heat exposure or chemicals.These wounds may be in normal individuals or those subject to conditionswhich induce abnormal wound healing such as diabetes, cornealdystrophies, uremia, malnutrition, vitamin deficiencies, obesity,infection, immunosuppression and complications associated with systemictreatment with steroids, radiation therapy, non-steroidalanti-inflammatory drugs (NSAID), anti-neoplastic drugs andanti-metabolites.

In certain embodiments, the present invention involves theadministration of pharmaceutical compositions that include galectin-3proteins with the amino acid sequence of human galectin-3 as representedby SEQ ID NO:1 of the sequence listing. In other embodiments, thepresent invention involves the administration of pharmaceuticalcompositions that include galectin-3 proteins with an amino acidsequence that is substantially identical to the amino acid sequence ofSEQ ID NO:1. For example, in certain embodiments, the present inventioninvolves the administration of pharmaceutical compositions that includegalectin-3 proteins which contain accidentally or deliberately inducedalterations, such as deletions, additions, substitutions ormodifications of the amino acid residues of SEQ ID NO:1. In yet otherembodiments, the present invention involves the administration ofpharmaceutical compositions that include proteins represented byfragments of the amino acid sequence SEQ ID NO:1 or hybrid proteins thatcomprise these fragments. Fragments of SEQ ID NO:1 preferably include agalectin-3 N-terminal domain and a galectin-3 proline, glycine, andtyrosine-rich domain; a galectin-3 proline, glycine, and tyrosine-richdomain and a galectin-3 galactoside-binding domain; or a galectin-3galactoside-binding domain.

In certain other embodiments, the present invention involves theadministration of pharmaceutical compositions that include galectin-7proteins with the amino acid sequence of human galectin-7 as representedby SEQ ID NO:2 of the sequence listing. In other embodiments, thepresent invention involves the administration of pharmaceuticalcompositions that include galectin-7 proteins with an amino acidsequence that is substantially identical to the amino acid sequence ofSEQ ID NO:2. For example, in certain embodiments, the present inventioninvolves the administration of pharmaceutical compositions that includegalectin-7 proteins which contain accidentally or deliberately inducedalterations, such as deletions, additions, substitutions ormodifications of the amino acid residues of SEQ ID NO:2. In yet otherembodiments, the present invention involves the administration ofpharmaceutical compositions that include proteins, represented byfragments of the amino acid sequence SEQ ID NO:2 or hybrid proteins thatcomprise these fragments. Preferred fragments of SEQ ID NO:2 include agalectin-7 galactoside-binding domain.

The present invention also encompasses the administration ofpharmaceutical compositions that include proteins represented by theamino acid sequence of galectin-3 and/or galectin-7 taken from anymammalian species including but not limited to bovine, canine, feline,caprine, ovine, porcine, murine, and equine species.

In certain embodiments, the pharmaceutical compositions of the presentinvention further include one or more additional therapeutic agents. Incertain embodiments, the additional therapeutic agent or agents areselected from the group consisting of growth factors, anti-inflammatoryagents, vasopressor agents, collagenase inhibitors, topical steroids,matrix metalloproteinase inhibitors, ascorbates, angiotensin II,angiotensin III, calreticulin, tetracyclines, fibronectin, collagen,thrombospondin, transforming growth factors (TGF), keratinocyte growthfactor (KGF), fibroblast growth factor (FGF), insulin-like growthfactors (IGF), epidermal growth factor (EGF), platelet derived growthfactor (PDGF), neu differentiation factor (NDF), hepatocyte growthfactor (HGF), and hyaluronic acid.

The pharmaceutical compositions of the present invention can beadministered to humans and other mammals topically, orally, rectally,parenterally, intracisternally, intravaginally, intraperitoneally,bucally, ocularly, or nasally, depending on the severity and location ofthe wound being treated. Administration may be therapeutic or it may beprophylactic. Liquid dosage forms for oral administration of aninventive pharmaceutical composition include, but are not limited to,pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. Solid dosage forms for oraladministration include capsules, tablets, pills, powders, and granules.Dosage forms for topical or transdermal administration includeointments, pastes, creams, lotions, gels, powders, solutions, sprays,inhalants, or patches. Injectable preparations may be in the form ofsterile injectable aqueous or oleaginous suspensions. Compositions forrectal or vaginal administration are preferably suppositories.Prophylactic formulations may be present or applied to the site ofpotential wounds, or to sources of wounds, such as contact lenses,contact lens cleaning and rinsing solutions, containers for contact lensstorage or transport, devices for contact lens handling, eye drops,surgical irrigation solutions, ear drops, eye patches, and cosmetics forthe eye area. The invention includes opthalmological devices, surgicaldevices, audiological devices or products which contain disclosedpharmaceutical compositions (e.g., gauze bandages or strips).

DESCRIPTION OF THE DRAWING

FIG. 1 depicts the amino acid sequence and composition of humangalectin-3 (Accession No. BAA22164 in GenBank, SEQ ID NO:1).

FIG. 2 depicts the amino acid sequence and composition of humangalectin-7 (Accession No. I55469 in GenBank, SEQ ID NO:2).

FIG. 3 depicts a CLUSTAL W alignment of the amino acid sequence of humangalectin-3 (SEQ ID NO:1) with the amino acid sequences of rabbitgalectin-3 (Accession No. JC4300 in GenBank), chicken galectin-3(Accession No. AAB02856 in GenBank), and hamster galectin-3 (AccessionNo. CAA55479 in GenBank). The first (upper) sequence in the figure isamino acids 1 to 250 of human galectin-3 (SEQ ID NO:1), the secondsequence in the figure is amino acids 1 to 245 of hamster galectin-3,the third sequence in the figure is amino acids 1 to 242 of rabbitgalectin-3, and the fourth (lower) sequence in the figure is amino acids1 to 262 of chicken galectin-3.

FIG. 4 depicts a CLUSTAL W alignment of the amino acid sequence of humangalectin-7 (SEQ ID NO:2) with the amino acid sequences of rat galectin-7(Accession No. P97590 in GenBank) and mouse galectin-7 (Accession No.O54974 in GenBank). The first (upper) sequence in the figure is aminoacids 1 to 136 of rat galectin-7, the second sequence in the figure isamino acids 1 to 136 of mouse galectin-7, and the third (lower) sequencein the figure is amino acids 1 to 136 of human galectin-7 (SEQ ID NO:2).

FIG. 5 is a summary of the results of a PROSITE scan of human galectin-3(SEQ ID NO:1).

FIG. 6 is a summary of the results of a PROSITE scan of human galectin-7(SEQ ID NO:2).

FIG. 7 depicts an alignment of the galactoside-binding domain of humangalectin-3 with a consensus amino acid sequence (PF00337) derived from ahidden Markov model (HMM) from PFAM. The upper sequence is the consensusamino acid sequence (PF00337, SEQ ID NO:3), while the lower amino acidsequence corresponds to amino acids 117 to 247 of SEQ ID NO:1.

FIG. 8 depicts an alignment of the galactoside-binding domain of humangalectin-7 with a consensus amino acid sequence (PF00337) derived from ahidden Markov model (HMM) from PFAM. The upper sequence is the consensusamino acid sequence (PF00337, SEQ ID NO:3), while the lower amino acidsequence corresponds to amino acids 5 to 135 of SEQ ID NO:2.

FIG. 9 includes a series of photographs of corneas with 2 mm abrasion orexcimer laser wounds that were allowed to partially heal in vivo andwere then analyzed for galectin-3 immunoreactivity in paraffin sections.(A), Hematoxylin and eosin staining of (i) normal corneas and corneasimmediately after (ii) abrasion and (iii) excimer laser injury. (B),Immunohistochemical staining of (i) normal gal3^(+/+) corneas and (ii)healing gal3^(+/+) corneas after excimer laser injury.Immunohistochemical staining of (iii) normal gal3^(−/−) corneas and (iv)healing gal3^(−/−) corneas after excimer laser injury. Dark colorindicates positive immunostaining. WE, wound edge; LE, leading edge ofmigrating epithelium; arrows, epithelium; arrowheads, leukocytes/stromalcells.

FIG. 10 is a graph illustrating the effect of β-lactose (Lac) andsucrose (Suc) on the healing rate of injured corneal epithelium.

FIG. 11 is a series of graphs illustrating the healing rate of injuredcorneal epithelium in wild type (gal-3^(+/+)) and galectin-3 deficient(gal-3^(−/−)) mice injured by excimer laser or alkali treatment andallowed to heal in vivo or in vitro.

FIG. 12 is a table depicting differences in gene expression ofgalectin-7 and a selection of house keeping genes (GAPDH isD-glyceraldehyde-3-phosphate dehydrogenase; RPS29 is ribosomal proteinS29; ODC is ornithine decarboxylase) between wild type (gal-3^(+/+)) andgalectin-3 deficient (gal-3^(−/−)) mice as determined by cDNA microarrayand semi-quantitative PCR.

FIG. 13 illustrates differences in the expression of galectin-7 betweenwild type (gal-3^(+/+)) and galectin-3 deficient (gal-3^(−/−)) mice asdetermined by (A) western blot analysis, (B) immunohistochemicalanalysis, and (C) using mouse embryonic fibroblasts.

FIG. 14 is a graph illustrating the effect of exogenous galectin-3 onthe healing rate of injured corneal epithelium in (A) galectin-3deficient (gal-3^(−/−)) mice and (B) wild type (gal-3^(+/+)) mice.

FIG. 15 is a graph illustrating the effect of β-lactose (Lac) andsucrose (Suc) on the healing rate of injured corneal epithelium of wildtype (gal-3^(+/+)) mice in the presence of exogenous galectin-3.

FIG. 16 includes (A) a graph illustrating the effect of exogenousgalectin-7 on the healing rate of injured corneal epithelium in wildtype (gal-3^(+/+)), when used alone, with β-lactose (Lac), or withsucrose (Suc); and (B) a graph comparing the effect of exogenousgalectin-7 on the healing rate of injured corneal epithelium in wildtype (gal-3^(+/+)) and galectin-3 deficient (gal-3^(−/−)) mice.

DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

The present application mentions various patents, scientific articles,and other publications. The contents of each such item are herebyincorporated by reference. In addition, the contents (as of the filingdate of the application) of all websites referred to herein areincorporated by reference.

The present invention provides pharmaceutical compositions comprisinggalectin-3 and/or galectin-7 useful for enhancing there-epithelialization of wounds in injured mammalian tissues. Theinvention also provides methods for the therapeutic treatment ofepithelial injuries in mammalian tissue comprising administering to amammal afflicted with an epithelial injury a therapeutically effectiveamount of galectin-3, galectin-7, or a combination of galectin-3 and -7.When administering a combination of galectin-3 and -7, galectin-3 may beadministered before, in conjunction with, or after the administration ofgalectin-7.

The invention encompasses the finding that galectin-3 is up-regulated inmigrating corneal epithelial cells following injury to the cornea(Example 1). The invention also includes the discovery that there-epithelialization of corneal transepithelial excimer laser wounds andcorneal alkali-burn wounds is significantly slower ingalectin-3-deficient mice compared to that in wild type mice (Example2). The invention further provides the discovery that the expression ofa number of injury-related genes (e.g., tolloid-like protein andgalectin-7) are abnormal in galectin-3-deficient mice (Example 3).Additionally, the invention demonstrates that exogenous galectin-3 and-7 promote the re-epithelialization of corneal wounds (Examples 4 and 5,respectively).

Galectins

Lectins are proteins that are defined by their ability to bindcarbohydrates specifically and to agglutinate cells (see, for example,Sharon, Trends Biochem. Sci. 18:221, 1993). Lectins have been shown tobe involved in a wide variety of cellular functions including cell-celland cell-matrix interactions. Lectins are widespread among plants,invertebrates and mammals. Animal lectins have been grouped into fourdistinct families: 1) C-type lectins; 2) P-type lectins; 3) galectins(formerly termed S-type lectins); and 4) pentraxins (see, for example,Barondes et al., J. Biol. Chem. 269:20807, 1994).

All mammalian galectins that have been analyzed in detail recognizeβ-lactose and related β-galactosides. While all mammalian galectinsshare similar affinity for small β-galactosides, they show significantdifferences in binding specificity for more complex glycoconjugates(Henrick et al., Glycobiology 8:45, 1998; Sato et al., J. Biol. Chem.267:6983, 1992; and Seetharaman et al., J. Biol. Chem. 273:13047, 1998).In addition to binding β-galactoside sugars, galectins possesshemagglutination activity. Laminin, a naturally occurring glycoproteincontaining numerous polylactosamine chains, has been shown to be anatural ligand for certain galectins. Laminin is a component of thebasal laminae, the extracellular matrix which underlies all epitheliaand surrounds individual muscle, fat and Schwann cells. Interactionsbetween cells and the basal laminae are known to influence the migrationand/or differentiation of various cell types during mammaliandevelopment. Galectins do not contain traditional sequences that specifymembrane translocation, but are both secreted and locatedintracellularly. In addition to their affinity for β-galactoside sugars,members of the galectin family share significant sequence similarity inthe carbohydrate recognition domain (CRD; also referred to as thecarbohydrate-binding domain), the relevant amino acid residues of whichhave been determined by X-ray crystallography (Lobsanov et al., J. Biol.Chem. 267:27034, 1993 and Seetharaman et al., supra). Galectins havebeen implicated in a wide variety of biological functions including celladhesion (Cooper et al., J. Cell Biol. 115:1437, 1991), growthregulation (Wells et al., Cell 64:91, 1991), cell migration (Hughes,Curr. Opin. Struct. Biol. 2:687, 1992), neoplastic transformation (Razet al., Int. J. Cancer 46:871, 1990) and immune responses (Offner etal., J. Neuroimmunol. 28:177, 1990). There are presently 12characterized eukaryotic members of the galectin family.

Galectin-3

Members of the galectin-3 family of proteins (previously known asCBP-35, Mac-2, L-34, εBP, and RL-29) typically include between about 240and 270 amino acids and have molecular weights that range between about25 and 29 kDa. Galectin-3 proteins are generally composed of a shortN-terminal domain, a C-terminal domain which includes agalactoside-binding region, and an intervening proline, glycine, andtyrosine-rich domain which includes repeats of 7-10 conserved aminoacids (Liu et al., Biochemistry 35:6073, 1996 and Cherayil et al., Proc.Natl. Acad. Sci. USA, 87:7324, 1990). The tandem repeats are similar tothose found in the collagen gene superfamily. The number of repeatsvaries between galectin-3 proteins and accounts for the differences insize between galectin-3 proteins from different species. The N-terminaldomain of galectin-3 permits the protein to undergo multimerization uponbinding to surfaces containing glycoconjugate ligands.

Galectin-3 is expressed in various inflammatory cells (e.g., activatedmacrophages, basophils, and mast cells) and in epithelia and fibroblastsof various tissues (Perillo et al., J. Mol. Med. 76:402, 1998). It isfound on the cell surface, within the extracellular matrix (ECM), in thecytoplasm, and in the nucleus of cells. On the cell surface or in theECM galectin-3 is thought to mediate cell-cell and cell-matrixinteractions by binding to complementary glycoconjugates containingpolylactosamine chains found in many ECM and cell surface molecules.Galectin-3 is thought to inhibit cell-matrix adhesion by binding tolaminin. In the nucleus of cells galectin-3 may influence cell-matrixinteractions indirectly by influencing the expression of well-known celladhesion molecules (e.g., α6β1 and α 4β7 integrins, Warlfield et al.,Invasion Metastasis 17:101, 1997 and Matarrese et al., Int. J. Cancer85:545, 2000) and cytokines (e.g., IL-1, Jeng et al., Immunol. Lett.42:113, 1994). Galectin-3 expression is developmentally regulated inselected organs such as the kidney and its expression level in pulmonaryalveolar epithelial cells and hepatocytes is up-regulated followinginjury. Galectin-3 has been shown to concentrate in the nucleus ofcertain cell types during proliferation. Expression of galectin-3 iselevated in certain tumors, suggesting galectin-3 plays a role inmetastasis. Indeed, overexpression of galectin-3 in a weakly metastaticcell line caused a significant increase in metastatic potential (Raz etal., supra).

Human galectin-3 is 250 amino acids long and has an approximatemolecular weight of 26.1 kDa (SEQ ID NO:1, FIG. 1). As illustrated inFIGS. 1, 3, 5, and 7, human galectin-3 contains the following domains,signature sequences, or other structural features (for generalinformation regarding PS and PF prefix identification numbers, refer toSonnhammer et al., Protein 28:405, 1997): an N-terminal domain locatedat about amino acid residues 1 to 14 of SEQ ID NO:1; a proline, glycine,and tyrosine-rich domain located at about amino acid residues 15 to 116of SEQ ID NO:1; a galactoside-binding domain located at about amino acidresidues 117 to 247 of SEQ ID NO:1; a galaptin signature sequence(PROSITE No. PS00309) located at about amino acids 181 to 200 of SEQ IDNO:1; one potential N-glycosylation site (PROSITE No. PS00001) locatedat about amino acids 4 to 7 of SEQ ID NO:1; two potential protein kinaseC phosphorylation sites (PROSITE No. PS00005) located at about aminoacids 137 to 139 and 194 to 196 of SEQ ID NO:1; two potential caseinkinase II phosphorylation sites (PROSITE No. PS00006) located at aboutamino acids 6 to 9 and 175 to 178 of SEQ ID NO: 1; and eight potentialmyristoylation sites (PROSITE No. PS00008) located at about amino acids24 to 29, 27 to 32, 34 to 39, 43 to 48, 52 to 57, 61 to 66, 65 to 70,and 68 to 73 of SEQ ID NO:1.

As defined herein, a “galectin-3 protein” may include a galectin-3“N-terminal domain”, a galectin-3 “proline, glycine, and tyrosine-richdomain”, and/or a galectin-3 “galactoside-binding domain”. These domainsare further defined as follows.

As used herein, a galectin-3 “N-terminal domain” includes an amino acidsequence of about 10-20 amino acids, preferably about 14 amino acidsthat shares at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100%identity with amino acids 1 to 14 of SEQ ID NO:1. The N-terminal domaincan include an N-glycosylation site (PROSITE No. PS00001) and/or acasein kinase II phosphorylation site (PROSITE No. PS00006). The PROSITEN-glycosylation site has the consensus sequence: N-{P}-[ST]-{P} and thePROSITE casein kinase II phosphorylation site has the consensussequence: [ST]-X(2)-[DE]. In the above consensus sequences, and othermotifs or signature sequences described herein, the standard IUPACone-letter code for the amino acids is used. Each element in the patternis separated by a dash (-); square brackets ([ ]) indicate theparticular residues that are accepted at that position; X indicates thatany residue is accepted at that position; and numbers in parentheses (()) indicate the number of residues represented by the accompanying aminoacid. In certain embodiments, the N-terminal domain includes amino acidsL7 and L11 of SEQ ID NO:1. As shown in FIG. 3, these amino acids areconserved across several mammalian species of galectin-3 and maytherefore play a catalytic and/or structural role.

As used herein, a galectin-3 “proline, glycine, and tyrosine-richdomain” includes an amino acid sequence of about 60 to 140 amino acids,more preferably about 80 to 120 amino acids, or about 90 to 110 aminoacids that shares at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100%identity with amino acids 15 to 116 of SEQ ID NO:1. The proline,glycine, and tyrosine-rich domain can also include one, two, three,four, five, six, seven, or eight N-myristoylation sites (PROSITE No.PS00008) which have the consensus sequence:G-{EDRKHPFYW}-X(2)-[STAGCN]-{P}. In certain embodiments, the proline,glycine, and tyrosine-rich domain includes the following amino acids andregions of SEQ ID NO:1: G21, P23, G27, N28, P30, G32, G34, P37, Y41-P46,G53, Y55-G57, P61, G62, G66, P72, G73, G77, Y79-G81, P83, G87, Y89, P90,G99, Y101, P102, P106, Y107, A109, L114, and V116. These amino acids andregions are conserved across several mammalian species of galectin-3 andmay play a catalytic and/or structural role (see amino acids indicatedwith a “*” in FIG. 3).

As used herein, a galectin-3 “galactoside-binding domain” includes anamino acid sequence of about 80 to 180 amino acids having a bit scorefor the alignment of the sequence to the consensus sequence PF00337 fromPFAM (SEQ ID NO:3) of at least 150. Preferably, a galectin-3galactoside-binding domain includes at least about 100 to 160 aminoacids, more preferably about 110 to 150 amino acids, or about 120 to 140amino acids and has a bit score for the alignment of the sequence to theconsensus sequence PF00337 from PFAM (SEQ ID NO:3) of at least 150, morepreferably at least 175, most preferably 200 or greater.

To calculate the bit score for the alignment of a particular sequence tothe consensus sequence PF00337 from PFAM, the sequence of interest canbe searched against the PFAM database of HMMs (e.g., the PFAM database,release 2.1) using the default parameters available atwww.sanger.ac.uk/Software/Pfam. A description of the PFAM database canbe found in Sonnhammer et al., supra and a detailed description of HMMscan be found, for example, in Gribskov et al., Meth. Enzymol. 183:146,1990 and Stultz et al., Protein Sci. 2:305, 1993.

The galectin-3 galactoside-binding domain can further include one,preferably two, protein kinase C phosphorylation sites (PROSITE No.PS00005); a casein kinase II phosphorylation site (PROSITE No. PS00006);and/or a galaptin signature sequence (PROSITE No. PS00309). The proteinkinase C phosphorylation site has the following consensus sequence:[ST]-X-[RK]. The galaptin signature sequence has the following consensussequence:W-[GEK]-X-[EQ]-X-[KRE]-X(3,6)-[PCTF]-[LIVMF]-[NQEGSKV]-X-[GH]-X(3)-[DENKHS]-[LIVMFC].In certain embodiments, the galectin-3 galactoside-binding domainincludes the following amino acids and regions of SEQ ID NO:1: P117,Y118, L120-L122, G125, P128, R129, L131-I134, G136-V138, N141, N143,R144, L147, F149, R151, G152, D154, A156-F163, E165, R169-N174,N179-G182, E184-R186, F190-E193, G195, P197-K199, Q201-L203, E205,D207-Q220, N222, R224, L228, I231, I236, G238-I240, and L242-S244. Theseamino acids and regions are conserved across several mammalian speciesof galectin-3 and may play a catalytic and/or structural role (see aminoacids indicated with a “*” in FIG. 3).

Certain galectin-3 proteins of the present invention include the aminoacid sequence of human galectin-3 as represented by SEQ ID NO:1. Othergalectin-3 proteins of the present invention include an amino acidsequence that is substantially identical to the amino acid sequence ofSEQ ID NO:1. The term “substantially identical” is used herein to referto a first amino acid that contains a sufficient or minimum number ofamino acid residues that are identical to aligned amino acid residues ina second amino acid sequence such that the first and second amino acidsequences can have a common structural domain and/or common functionalactivity. For example, amino acid sequences that contain a commonstructural domain having at least about 60%, or 65% identity, preferablyat least 75% identity, more preferably at least 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1 are termedsubstantially identical to the amino acid sequence of SEQ ID NO:1. Inparticular, proteins which contain accidentally or deliberately inducedalterations, such as deletions, additions, substitutions ormodifications of certain amino acid residues of SEQ ID NO:1 may fallwithin the definition of galectin-3 proteins provided herein. It willalso be appreciated that as defined herein, galectin-3 proteins mayinclude regions represented by the amino acid sequence of galectin-3taken from other mammalian species including but not limited to bovine,canine, feline, caprine, ovine, porcine, murine, and equine species.

Calculations of sequence identity between sequences are performed asfollows. To determine the percent identity of two amino acid sequences,the sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acidsequence for optimal alignment). The amino acid residues atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the proteins are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using an alignment software programusing the default parameters. Suitable programs include, for example,CLUSTAL W by Thompson et al., Nuc. Acids Research 22:4673, 1994(www.ebi.ac.uk/clustalw), BL2SEQ by Tatusova and Madden, FEMS Microbiol.Lett. 174:247, 1999 (www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html), SAGAby Notredame and Higgins, Nuc. Acids Research 24:1515, 1996(igs-server.cnrs-mrs.fr/˜cnotred), and DIALIGN by Morgenstern et al.,Bioinformatics 14:290, 1998 (bibiserv.techfak.uni-bielefeld.de/dialign).

Galectin-7

Members of the galectin-7 family of proteins typically exist as monomersthat include between about 130 and 140 amino acids and have molecularweights that range between about 15 and 16 kDa (see, for example,Magnaldo et al., Develop. Biol. 168:259, 1995 and Madsen et al., J.Biol. Chem. 270:5823, 1995). The expression of galectin-7 has beenassociated with the onset of epithelial stratification (Timmons et al.,Int. J. Dev. Biol. 43:229, 1999). Galectin-7 is thought to play a rolein cell-matrix and cell-cell interactions. Galectin-7 is found in areasof cell-cell contact (e.g., in the upper layers of human epidermis); itsexpression is sharply downregulated in anchorage independentkeratinocytes and it is absent in a malignant keratinocyte cell line.Galectin-7 may be required for the maintenance of normal keratinocytes(see, Madsen et al., supra).

Human galectin-7 includes 136 amino acids and has an approximatemolecular weight of 15.1 kDa (SEQ ID NO:2, FIG. 2). As illustrated inFIGS. 2, 4, 6, and 8, human galectin-7 contains the following domains,signature sequences, or other structural features: a galactoside-bindingdomain located at about amino acid residues 5 to 135 of SEQ ID NO:2; agalaptin signature sequence (PROSITE No. PS00309) located at about aminoacids 70 to 89 of SEQ ID NO:2; one N-glycosylation site (PROSITE No.PS00001) located at about amino acids 29 to 32 of SEQ ID NO:2; oneprotein kinase C phosphorylation site (PROSITE No. PS00005) located atabout amino acids 132 to 134 of SEQ ID NO:2; one casein kinase IIphosphorylation site (PROSITE No. PS00006) located at about amino acids9 to 12 of SEQ ID NO:2; and two myristoylation sites (PROSITE No.PS00008) located at about amino acids 13 to 18 and 44 to 49 of SEQ IDNO:2.

As defined herein, a “galectin-7 protein” includes a galectin-7“galactoside-binding domain”. This domain is further defined as follows.

As used herein, a galectin-7 “galactoside-binding domain” includes anamino acid sequence of about 80 to 180 amino acids having a bit scorefor the alignment of the sequence to the consensus sequence PF00337 fromPFAM (SEQ ID NO:3) of at least 80. Preferably, a galectin-7galactoside-binding domain includes at least about 100 to 160 aminoacids, more preferably about 110 to 150 amino acids, or about 120 to 140amino acids and has a bit score for the alignment of the sequence to theconsensus sequence PF00337 from PFAM (SEQ ID NO:3) of at least 80, morepreferably at least 100, most preferably 120 or greater. The galectin-7galactoside-binding domain can include one N-glycosylation site (PROSITENo. PS00001); one protein kinase C phosphorylation site (PROSITE No.PS00005); one casein kinase II phosphorylation site (PROSITE No.PS00006); one or two myristoylation sites (PROSITE No. PS00008); and/ora galaptin signature sequence (PROSITE No. PS00309). In certainembodiments, the galectin-7 galactoside-binding domain includes thefollowing amino acids and regions of SEQ ID NO:2: M1, S2, H6, K7, L10,P11, G13, R15, G17-V19, R21-G24, V26, P27, A30, R32-Q43, D46-N63, K65,Q67, G68, W70-G76, G78, P80-L90, I92, G97-K99, V101, G103, D104, Y107,H109, F110, H112, R113, P115, V119, R120, V122-L130, S132, I135, andF136. These amino acids and regions are conserved across severalmammalian species of galectin-7 and may play a catalytic and/orstructural role (see amino acids indicated with a“*” in FIG. 4).

Certain galectin-7 proteins of the present invention include the aminoacid sequence of human galectin-7 as represented by SEQ ID NO:2. Othergalectin-7 proteins of the present invention include an amino acidsequence that is substantially identical to the amino acid sequence ofSEQ ID NO:2. In particular, proteins which contain accidentally ordeliberately induced alterations, such as deletions, additions,substitutions or modifications of certain amino acid residues of SEQ IDNO:2 may fall within the definition of galectin-7 provided herein. Itwill also be appreciated that as defined herein, galectin-7 proteins mayinclude regions represented by the amino acid sequence of galectin-7taken from other mammalian species including but not limited to bovine,canine, feline, caprine, ovine, porcine, murine, and equine species.

Preparation of Galectin-3 and Galectin-7

It will be appreciated by one of ordinary skill in the art, that thegalectins of this invention can be obtained from any available source.These include but are not limited to proteins isolated from naturalsources, produced recombinantly or produced synthetically, e.g., bysolid phase procedures. In accordance with the present invention,polynucleotide sequences which encode galectin-3 or galectin-7 may beused in recombinant DNA molecules that direct the expression of thegalectins of this invention in appropriate host cells. Cherayil et al.,supra and Madsen et al., supra, describe in detail the cloning of humangalectin-3 and -7 respectively. In order to express a biologicallyactive galectin-3 or galectin-7, the nucleotide sequence encodinggalectin-3, galectin-7, or their functional equivalent, is inserted intoan appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart can be used to construct expression vectors containing agalectin-3-encoding or galectin-7-encoding sequence and appropriatetranscriptional or translational controls. These methods include invitro recombinant DNA techniques, synthetic techniques and in vivorecombination or genetic recombination. The introduction of deletions,additions, or substitutions can be achieved using any known technique inthe art e.g., using PCR based mutagenisis. Such techniques are describedin Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, Plainview, N.Y., 1989 and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1989.A variety of expression vector/host systems may be utilized to containand express a galectin-3-encoding or galectin-7-encoding sequence. Theseinclude but are not limited to microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.,baculovirus); plant cell systems transfected with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with bacterial expression vectors (e.g., Ti, pBR322,or pET25b plasmid); or animal cell systems. Alternatively, the galectinsof the present invention could be produced using chemical methods tosynthesize a galectin-3 or galectin-7 amino acid sequence, whole or inpart. For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge et al., Science 269:202, 1995) andautomated synthesis may be achieved, for example, using the 431A peptidesynthesizer (available from Applied Biosystems of Foster City, Calif.)in accordance with the instructions provided by the manufacturer.

Pharmaceutical Compositions

In one aspect of the present invention, pharmaceutical compositions areprovided, wherein these compositions comprise galectin-3 and/orgalectin-7, and optionally comprise a pharmaceutically acceptablecarrier. In certain embodiments, these compositions optionally furthercomprise one or more additional therapeutic agents. In certainembodiments, the additional therapeutic agent or agents are selectedfrom the group consisting of growth factors, anti-inflammatory agents,vasopressor agents, collagenase inhibitors, topical steroids, matrixmetalloproteinase inhibitors, ascorbates, angiotensin II, angiotensinIII, calreticulin, tetracyclines, fibronectin, collagen, thrombospondin,transforming growth factors (TGF), keratinocyte growth factor (KGF),fibroblast growth factor (FGF), insulin-like growth factors (IGF),epidermal growth factor (EGF), platelet derived growth factor (PDGF),neu differentiation factor (NDF), hepatocyte growth factor (HGF), andhyaluronic acid.

As used herein, the term “pharmaceutically acceptable carrier” includesany and all solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences Ed. by Gennaro, Mack Publishing, Easton, Pa.,1995 discloses various carriers used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof. Someexamples of materials which can serve as pharmaceutically acceptablecarriers include, but are not limited to, sugars such as glucose, andsucrose; starches such as corn starch and potato starch; cellulose andits derivatives such as sodium carboxymethyl cellulose, ethyl cellulose,and cellulose acetate; powdered tragacanth; malt; gelatin; talc;excipients such as cocoa butter and suppository waxes; oils such aspeanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, cornoil, and soybean oil; glycols; such a propylene glycol; esters such asethyl oleate and ethyl laurate; agar; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe composition, according to the judgment of the formulator.

Therapeutically Effective Dose

In yet another aspect, according to the methods of treatment of thepresent invention, the re-epithelialization of wounds is promoted bycontacting the wounds with a pharmaceutical composition, as describedherein. Thus, the invention provides methods for the treatment of woundscomprising administering a therapeutically effective amount of apharmaceutical composition comprising active agents that includegalectin-3 and/or galectin-7 to a subject in need thereof, in suchamounts and for such time as is necessary to achieve the desired result.It will be appreciated that this encompasses administering an inventivepharmaceutical as a therapeutic measure to promote there-epithelialization of a wound or as a prophylactic measure to minimizecomplications associated with the slow re-epithelialization of wounds(e.g., as a wound irrigation solution during and/or following surgery).In certain embodiments of the present invention a “therapeuticallyeffective amount” of the pharmaceutical composition is that amounteffective for promoting the re-epithelialization of a wound. Thecompositions, according to the method of the present invention, may beadministered using any amount and any route of administration effectivefor healing a wound. Thus, the expression “amount effective forpromoting the re-epithelialization of a wound”, as used herein, refersto a sufficient amount of composition to heal a wound. The exact dosageis chosen by the individual physician in view of the patient to betreated. Dosage and administration are adjusted to provide sufficientlevels of the active agent(s) or to maintain the desired effect.Additional factors which may be taken into account include the severityof the disease state, e.g., wound size and location; age, weight andgender of the patient; diet, time and frequency of administration; drugcombinations; reaction sensitivities; and tolerance/response to therapy.Long acting pharmaceutical compositions might be administered every 3 to4 days, every week, or once every two weeks depending on half-life andclearance rate of the particular composition.

The active agents of the invention are preferably formulated in dosageunit form for ease of administration and uniformity of dosage. Theexpression “dosage unit form” as used herein refers to a physicallydiscrete unit of active agent appropriate for the patient to be treated.It will be understood, however, that the total daily usage of thecompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. For any activeagent, the therapeutically effective dose can be estimated initiallyeither in cell culture assays or in animal models, usually mice,rabbits, dogs, or pigs. The animal model is also used to achieve adesirable concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. A therapeutically effective dose refers tothat amount of active agent which ameliorates the symptoms or condition.Therapeutic efficacy and toxicity of active agents can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED50 (the dose is therapeutically effective in 50% of thepopulation) and LD50 (the dose is lethal to 50% of the population). Thedose ratio of toxic to therapeutic effects is the therapeutic index, andit can be expressed as the ratio, LD50/ED50. Pharmaceutical compositionswhich exhibit large therapeutic indices are preferred. The data obtainedfrom cell culture assays and animal studies is used in formulating arange of dosage for human use.

Administration of Pharmaceutical Compositions

After formulation with an appropriate pharmaceutically acceptablecarrier in a desired dosage, the pharmaceutical compositions of thisinvention can be administered to humans and other mammals topically (asby powders, ointments, or drops), orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, bucally, ocularly,or nasally, depending on the severity and location of the wound beingtreated.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active agent(s), theliquid dosage forms may contain inert diluents commonly used in the artsuch as, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Dosage forms for topical or transdermal administration of an inventivepharmaceutical composition include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants, or patches. The activeagent is admixed under sterile conditions with a pharmaceuticallyacceptable carrier and any needed preservatives or buffers as may berequired. For example, ocular or cutaneous infections may be treatedwith aqueous drops, a mist, an emulsion, or a cream. Administration maybe therapeutic or it may be prophylactic. Prophylactic formulations maybe present or applied to the site of potential wounds, or to sources ofwounds, such as contact lenses, contact lens cleaning and rinsingsolutions, containers for contact lens storage or transport, devices forcontact lens handling, eye drops, surgical irrigation solutions, eardrops, eye patches, and cosmetics for the eye area, including creams,lotions, mascara, eyeliner, and eyeshadow. The invention includesopthalmological devices, surgical devices, audiological devices orproducts which contain disclosed compositions (e.g., gauze bandages orstrips), and methods of making or using such devices or products. Thesedevices may be coated with, impregnated with, bonded to or otherwisetreated with a disclosed composition.

The ointments, pastes, creams, and gels may contain, in addition to anactive agent of this invention, excipients such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to the agents of thisinvention, excipients such as talc, silicic acid, aluminum hydroxide,calcium silicates, polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants such aschlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of the active ingredients to the body. Such dosage forms can bemade by dissolving or dispensing the compound in the proper medium.Absorption enhancers can also be used to increase the flux of thecompound across the skin. The rate can be controlled by either providinga rate controlling membrane or by dispersing the compound in a polymermatrix or gel.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Theinjectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use. In order to prolong the effect of an active agent, it is oftendesirable to slow the absorption of the agent from subcutaneous orintramuscular injection. Delayed absorption of a parenterallyadministered active agent may be accomplished by dissolving orsuspending the agent in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the agent in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofactive agent to polymer and the nature of the particular polymeremployed, the rate of active agent release can be controlled. Examplesof other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the agent in liposomes or microemulsions which are compatiblewith body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the active agent(s) ofthis invention with suitable non-irritating excipients or carriers suchas cocoa butter, polyethylene glycol or a suppository wax which aresolid at ambient temperature but liquid at body temperature andtherefore melt in the rectum or vaginal cavity and release the activeagent(s).

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeagent is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, sucrose, glucose,mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as milksugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings, release controlling coatings and other coatings well known inthe pharmaceutical formulating art. In such solid dosage forms theactive agent(s) may be admixed with at least one inert diluent such assucrose or starch. Such dosage forms may also comprise, as is normalpractice, additional substances other than inert diluents, e.g.,tableting lubricants and other tableting aids such a magnesium stearateand microcrystalline cellulose. In the case of capsules, tablets andpills, the dosage forms may also comprise buffering agents. They mayoptionally contain opacifying agents and can also be of a compositionthat they release the active agent(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes.

Uses of Pharmaceutical Compositions

As discussed above and described in greater detail in the Examples,galectin-3 and galectin-7 are useful as promoters of there-epithelialization of wounds. In general, it is believed that thesegalectins will be clinically useful in stimulating the healing of woundsassociated with any epithelial tissue including but not limited to theskin epithelium; the corneal epithelium; the lining of thegastrointestinal tract; the lung epithelium; and the inner surface ofkidney tubules, of blood vessels, of the uterus, of the vagina, of theurethra, or of the respiratory tract. The present invention encompassesthe treatment of a variety of epithelial wound types including but notlimited to surgical wounds, excisional wounds, blisters, ulcers,lesions, abrasions, erosions, lacerations, boils, cuts, sores, and burnsresulting from heat exposure or chemicals. These wounds may be in normalindividuals or those subject to conditions which induce abnormal woundhealing such as diabetes, corneal dystrophies, uremia, malnutrition,vitamin deficiencies, obesity, infection, immunosuppression andcomplications associated with systemic treatment with steroids,radiation therapy, non-steroidal anti-inflammatory drugs (NSAID),anti-neoplastic drugs and anti-metabolites.

Galectin-3 and/or galectin-7 could, for example, be used to promotedermal re-establishment subsequent to dermal loss. Alternatively,galectin-3 and/or galectin-7 could be used to increase the adherence ofskin grafts to a wound bed and to stimulate re-epithelialization fromthe wound bed. Suitable skin grafts include, but are not limited to,autografts, artificial skin, allografts, autodermic grafts,autoepidermic grafts, avacular grafts, Blair-Brown grafts, bone grafts,brephoplastic grafts, cutis grafts, delayed grafts, dermic grafts,epidermic grafts, fascia grafts, full thickness grafts, heterologousgrafts, xenografts, homologous grafts, hyperplastic grafts, lamellargrafts, mesh grafts, mucosal grafts, Ollier-Thiersch grafts, omenpalgrafts, patch grafts, pedicle grafts, penetrating grafts, split skingrafts, and thick split grafts.

Galectin-3 and/or galectin-7 could be used to treat dermatitisherpetiformis in which blisters form at the dermo-epidermal junction.Galectin-3 and/or galectin-7 could be used to treat epidermolysisbullosa, a defect in adherence of the epidermis to the underlying dermiswhich results in frequent, open and painful blisters, by acceleratingre-epithelialization of these lesions. Galectin-3 and/or galectin-7could further be used to treat pemphigus diseases that involve loss ofcell-cell adhesion within the epidermis, or pemphigoid diseases thatinvolve loss of cell-cell adhesion at the dermo-epidermal junction.Galectin-3 and/or galectin-7 could be used to treat a variety of ulcersincluding but not limited to diabetic ulcers, dermal ulcers, decubitusulcers, arterial ulcers, and venous stasis ulcers.

The present invention encompasses methods for the promotion of cornealtissue healing. This includes treating corneal epithelial defects causedby corneal ulcers, heat, radiation, phlyctenulosis, corneal abrasions orlacerations, photorefractive surgery for corrective myopia, foreignbodies and sterile corneal infiltrates; chemical burns caused byexposure to acids or alkali (e.g., hydrofluoric acid, formic acid,anhydrous ammonia, cement, and phenol) or other chemical agents such aswhite phosphorus, elemental metals, nitrates, hydrocarbons, and tar;keratopathies such as neurotrophic keratopathy, diabetic keratopathy andThygeson's superficial punctate keratopathy; keratities such as viralkeratitis (e.g., metaherpetic or herpetic keratitis) and bacterialkeratitis; and corneal dystrophies such as lattice dystrophy, epithelialbasement membrane dystrophy (EBMD) and Fuch's endothelial dystrophy.

Galectin-3 and/or galectin-7 could also be used to treatgastrointestinal ulcers and help heal the mucosal lining andregeneration of glandular mucosa and duodenal mucosal lining morerapidly. Inflammatory bowel diseases, such as Crohn's disease andulcerative colitis, are diseases which result in destruction of themucosal surface of the small or large intestine, respectively. Thus,galectin-3 and galectin-7 could be used to promote the resurfacing ofthe mucosal surface to aid more rapid healing and to prevent orattenuate progression of inflammatory bowel disease. Galectin-3 andgalectin-7 would be expected to bind mucin and facilitate its adhesionto the apical surface of the epithelium and could therefore be used toprotect the gastrointestinal tract from injurious substances that areingested or following surgery. Galectin-3 and/or galectin-7 could beused to reduce the side effects of gut toxicity that result from thetreatment of bacterial infections, viral infections, radiation therapy,chemotherapy or other treatments. Galectin-3 and/or galectin-7 may, forexample, be used prophylactically or therapeutically to prevent orattenuate mucositis, esophagitis, or gastritis (e.g., to heal lesionsassociated with oral, esophageal, intestinal, colonic, rectal, and analulcers).

Galectin-3 and/or galectin-7 could be used to promote urothelialhealing. Tissue layers comprising urothelial cells may be damaged bynumerous mechanisms including catheterization, surgery, or bacterialinfection (e.g., infection by an agent which causes a sexuallytransmitted disease, such as gonorrhea). The present invention alsoencompasses methods for the promotion of tissue healing in the femalegenital tract comprising the administration of an effective amount ofgalectin-3 and/or galectin-7. Tissue damage in the female genital tractmay be caused by a wide variety of conditions including Candidainfections trichomoniasis, Gardnerella, gonorrhea, chlamydia, mycoplasmainfections and other sexually transmitted diseases.

Galectin-3 and/or galectin-7 could be used to promote the repair ofrenal epithelial cells and, thus, could be useful for alleviating ortreating renal diseases and pathologies such as acute and chronic renalfailure and end stage renal disease. Galectin-3 and/or galectin-7 couldbe used to promote the repair of breast tissue and therefore could beused to promote healing of breast tissue injury due to surgery, trauma,or cancer. Galectin-3 and/or galectin-7 could further be used to promotehealing and alleviate damage of brain tissue due to injury from trauma,surgery or chemicals.

Galectin-3 and/or galectin-7 could be administered prophylactically toreduce or prevent damage to the lungs caused by various pathologicalstates. For example, galectin-3 and/or galectin-7 could be used topromote the repair of alveoli and bronchiolar epithelium to prevent,attenuate, or treat acute or chronic lung damage. Emphysema, whichresults in the progressive loss of alveoli, and inhalation injuries,i.e., resulting from smoke inhalation and burns, that cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively treatedusing galectin-3 and/or galectin-7 as could damage attributable tochemotherapy, radiation treatment, lung cancer, asthma, black lung andother lung damaging conditions.

It will be appreciated that the therapeutic methods encompassed by thepresent invention are not limited to treating wounds in humans, but maybe used to treat wounds in any mammal including but not limited tobovine, canine, feline, caprine, ovine, porcine, murine, and equinespecies. When treating wounds in a given species, it is preferred, butnot required, that the galectin-3 and/or galectin-7 used, have an aminoacid sequence that is substantially identical to the amino acid sequenceof galectin-3 and/or galectin-7 as it occurs naturally in said species.

EXAMPLES

All animal treatments described in these examples conformed to theAssociation for Research in Vision and Opthalmology Resolution on theUse of Animals in Vision Research and the recommendations of the NIHGuide for the Care and Use of Laboratory Animals.

Example 1 Up-Regulation of Galectin-3 in Migrating Corneal EpitheliumFollowing Injury

To determine whether the expression level of galectin-3 is altered inthe epithelium of healing corneas following injury, mice corneas with 2mm excimer laser ablations and abrasion wounds, were allowed topartially heal in vivo and were then processed for immunostaining withrat anti-human galectin-3 mAb M3/38 (American Type Culture Collection,Rockville, Md.). Corneal epithelium is a prototype-stratified squamousepithelium. In mouse, it constitutes 20-25% of total corneal thicknessand is composed of 5 to 6 layers of cells. Posterior to the epithelialbasement membrane is corneal stroma, which in mouse represents 70-80% ofthe total corneal thickness. Abrasion wounds remove epithelium leavingthe corneal stroma intact. In contrast, excimer laser treatment, whichis commonly used for correction of myopia, removes epithelium as well asanterior corneal stroma.

Swiss Webster mice (Taconic Laboratory Animal Services, Germantown,N.Y.) were anesthetized by an intramuscular injection of 1.25% avertin(0.2 ml/10 kg body weight). Avertin was prepared by mixing 2.5 g of2,2,2 tribromoethanol, 5 ml 2-methyl-2-butanol (Aldrich, Milwaukee,Wis.) and 195 ml distilled water. Proparacaine eye drops (ALCAINE™available from Alcon Labs, Fort Worth, Tex.) were applied to the corneaas topical anesthetic. Transepithelial excimer laser ablations wereperformed on the right eyes of a first group of mice (2 mm optical zone;42 to 44 μm ablation depth, PTK mode) using an APEX PLUS™ excimer laser(Summit Technology of Waltham, Mass.). 2 mm abrasion wounds wereproduced on the right eyes of a second group of mice using an Algerbrush (Alger Equipment Company of Lago Vista, Tex.).

Following surgery, all animals received an intramuscular injection ofbuprenorphine (0.2 ml of 0.3 mg/ml, BUPRENEX™ available from Reckitt &Colman Pharmaceuticals, Richmond, Va.) as a pain killer. Antibioticointment (VETROPOLYCIN™ available from Pharmaderm, Melville, N.Y.) wasapplied and the corneas were allowed to partially heal in vivo for 16 to18 hours. At the end of the healing period the animals were anesthetizedas described above and were sacrificed by cervical dislocation. The eyeswere then fixed in formalin for two hours prior to embedding in paraffinwax. Tissue sections (5 μm thick) were cut in the place parallel to theocular axis. The sections were deparaffinized by treatment with xylineand re-hydrated with graded ethanol solutions (100%, 70%, and 30%). Forimmunostaining, tissue sections were incubated sequentially with 3% H₂O₂(37° C., 10 min), and 2.5% normal goat serum to block endogenousperoxidase activity and nonspecific binding, respectively. The sectionswere subsequently incubated with mAb M3/38 (undiluted hybridoma fluid, 1hour), biotinylated anti-rat IgG for 1 hour (1:200, Vector Labs,Burlingame, Calif.), a freshly prepared complex of avidin D andbiotinperoxidase for 20 hours (Vector Labs) and diaminobenzidine(DAB)—H₂O₂ reagent (Kirkegaard & Perry Labs, Gaithersburg, Md.). Fornegative controls, sections were treated with an irrelevant mAb or mediaalone.

As shown in FIG. 9, immunohistochemical staining of paraffin sections ofnormal (FIGS. 9 A and B) and healing (FIGS. 9 C and D) corneas indicatedthat in both models of corneal wound healing, the leading edge of themigrating epithelium of healing corneas stained more intensely with mAbM3/38 compared to the normal epithelium, especially in the basal andmiddle cell layers. In both healing as well as normal cornealepithelium, immunostaining was more intense at the site of cell-matrixattachment. While stromal cells of normal corneas did not react with mAbM3/38, cells in the anterior stroma under the healing corneas robustlyexpressed galectin-3, especially in the region under the migratingepithelium.

The galectin-3 immunoreactivity in corneal epithelium was similar whencorneas were allowed to heal in serum-free Eagle's minimum essentialmedium containing nonessential amino acids, L-glutamine, antibiotics and0.4% bovine serum albumin (BSA) in organ culture for 16 to 18 hours.However, anterior stroma of corneas that were allowed to heal in vitrolacked cells expressing galectin-3, suggesting that the galectin-3positive cells seen in the stroma of corneas that were allowed to healin vivo are most likely leukocytes and not keratocytes.

To determine whether the carbohydrate recognition domain of galectin-3plays a role in corneal epithelial sheet migration following injury,corneas with 2 mm excimer laser and abrasion wounds were allowed to healin organ culture in the presence and absence of the disaccharidesβ-lactose and sucrose. While β-lactose contains galactose and bindsgalectins, sucrose lacks galactose and does not bind galectins. In theseexperiments, the rate of re-epithelialization of corneal wounds wassignificantly slower in the presence of β-lactose, while sucrose had noeffect. As shown in FIG. 10, healing rates expressed as mm²/h among thedifferent groups (mean±SEM of at least two experiments) were: mediaalone, 0.088±0.003 (N=29); media plus β-lactose, 0.063±0.003 (N=19);media plus sucrose 0.084±0.004 (N=10).

Example 2 Corneal Epithelial Wound Closure in Wild Type and Galectin-3Deficient Mice

To determine whether the re-epithelialization of corneal wounds isimpaired in galectin-3 deficient mice, four different models of cornealwound healing were used. Galectin-3 deficient mice (gal-3^(−/−)) weregenerated by targeted interruption of the galectin-3 gene as describedin Hsu et al., Am. J. Pathol. 156:1073, 2000. Specifically, the regioncoding for the CRD was interrupted with a neomycin resistant gene. Thisinvolved substituting a 0.5 kb intron 4-exon 5 segment with theantibiotic resistant gene (neo). That the galectin-3 gene has beeninactivated was confirmed by Southern blot as well as Western blotanalysis.

Briefly, corneas with excimer laser ablations (as described inExample 1) or alkali-burn wounds were allowed to partially heal in vivoor in vitro (as described in Example 1). For alkali injury, 2 mm filterdiscs (Whatman 50, Whatman International, Maidstone, UK) were preparedusing a trephine, soaked in 0.5N NaOH, and placed on the surface of thecornea of the right eyes of a second group of mice for 30 seconds. Theeyes were then rinsed with excess PBS. At the end of the healing period,the wound areas were visualized by staining with methylene blue. Thestained wounds were then photographed at a standard distance, and theoutlines of the wound areas were traced on paper from projected imagesof the stained wounds. These outlines were digitized and quantifiedusing SIGMASCAN™ software (SPSS Science of Chicago, Ill.). Analysis ofthe wound closure rate in gal-3^(+/+) mice in different models of woundhealing revealed that wound closure rate expressed as mm²/h ingal-3^(+/+) mice was slower in corneas injured with an excimer lasercompared to those injured with an alkali-burn. Also, regardless of theinjury method used, the wound closure rate was faster in corneas allowedto heal in vivo compared to those in organ culture. As shown in FIG. 11,wound closure rates among gal-3^(+/+) groups were: 0.076±0.003 mm²/h forthe excimer laser/in vivo group, 0.050±0.003 mm²/h for the excimerlaser/in vitro group, 0.182±0.003 mm²/h for the alkali-burn/in vivogroup, and 0.106±0.005 mm²/h for the alkali-burn/in vitro group. Eachgroup represents the mean±SEM of at least two experiments (N=9 or morein each group). Comparison of the wound closure rate of gal-3^(+/+)groups with gal-3^(−/−) groups revealed that regardless of whether thecorneas were injured by excimer laser or by alkali treatment and whetherthe corneas were allowed to heal in vivo or in vitro, corneal epithelialwound closure rate expressed in mm²/h was significantly slower in thegal-3^(−/−) mice compared to that in the gal-3^(+/+) mice. Wound closurerates among different gal-3^(−/−) groups were 0.060±0.004 mm²/h for theexcimer laser/in vivo group, 0.036±0.005 mm²/h for the excimer laser/invitro group, 0.150±0.008 mm² for the alkali-burn/in vivo group, and0.081±0.004 mm²/h for the alkali-burn/in vitro group. Again, all valuesare the mean±SEM of at least two experiments (N=8 or more in eachgroup).

Example 3 Gene Expression Patterns in Migrating Corneal Epithelium ofGalectin-3 Deficient Mice Following Injury

In an attempt to understand why the re-epithelialization of cornealepithelial wounds is perturbed in gal-3^(−/−) mice, gene expressionpatterns of healing gal-3^(+/+) and gal-3^(−/−) corneas were comparedusing cDNA microarrays and the results were further confirmed bysemiquantitative RT-PCR.

Transepithelial excimer laser ablations (2 mm diameter) were produced onthe right eye of 30 gal^(+/+) and 30 gal^(−/−) mice as described inExample 1. Corneas were allowed to partially heal in vivo for 20 to 24hours. At the end of the healing period, animals were sacrificed and thecorneas were excised and immediately placed in liquid nitrogen andshipped to Clontech Laboratories, Palo Alto, Calif. for analysis of geneexpression using SMART™ cDNA technology. Briefly, total RNA was isolatedusing the reagents provided in the ATLAS™ Pure Total RNA LabelingSystem. Yield of RNA from the 30 gal-3^(+/+) and 30 gal-3^(−/−) corneaswas 3.5 μg and 2.6 μg respectively. The A260:A280 ratio of the RNApreparations of the corneas of gal-3^(+/+) and gal-3^(−/−) mice were1.48 and 1.37 respectively. The ribosomal RNA 28S:18S ratio was 1.8 forboth preparations. This ensured that the quality of RNA preparation wassatisfactory. For probe preparation, first strand cDNA was synthesizedusing 175 ng of RNA, a modified oligo(dT) primer (the CDS primer),POWERSCRIPT™ reverse transcriptase, and SMART™ II oligonucleotides.Controls involved incubation of samples without reverse transcriptase.The cDNA was amplified by long distance (LD)-PCR. To determine theoptimal number of amplification cycles, aliquots of reaction productswere collected at 15, 18, 21 and 24 cycles and were analyzed by agarosegel electrophoresis. The yield of amplified double stranded cDNA usingan optimal number of cycles, i.e., 23, was between 1 and 1.64 μg. Theamplified cDNAs (500 ng) were radiolabeled using Klenow enzyme and³³P-αATP as described in the instruction manual for SMART™ cDNA probesynthesis for ATLAS™ microarrays (Clontech). The labeled probes werepurified by filtration on a NUCLEOSPIN™ filter and were then hybridizedto mouse 1.2k-I ATLAS™ nylon cDNA microarrays (Clontech). This is abroad spectrum array consisting of ˜1200 mouse genes. Followinghybridization, the membranes were exposed to a phosphorimager screen andthe results were analyzed by ATLAS IMAGE™ 2.0 software (Clontech). Thedata were verified by semiquantitative RT-PCR.

For RT-PCR, total RNA and first strand cDNA were prepared from healinggal-3^(+/+) and gal-3^(−/−) corneas using the procedures describedearlier. PCR amplification was performed in 50 μl volume using 14 ng ofcDNA, gene-specific custom primers purchased from Clontech and otherreagents from the ADVANTAGE™ 2 PCR kit (Clontech). The annealingtemperature used was 68° C. and reactions were subjected to varyingnumber of cycles of PCR amplification. For analysis of housekeepinggenes, 5 μl aliquots of amplified product were collected at every 5^(th)cycle, beginning at the 18^(th) cycle, whereas for analysis ofdifferentially expressed genes reaction amplified products werecollected at every other cycle, beginning at the 28^(th) cycle.Amplified products collected at various cycles were analyzed byelectrophoresis in 1.5% agarose/ethedium bromide gels (FIG. 12).

These experiments revealed that compared to healing corneas ofgal-3^(+/+) mice, healing corneas of gal-3^(−/−) mice contain markedlyreduced levels of mRNA transcripts for galectin-7, anothergalactose-binding protein, and tolloid-like protein (TLL), ametalloproteinase. Overall, compared to healing gal-3^(+/+) corneas,healing gal-3^(−/−) corneas contained about 12 times less galectin-7(FIG. 12) and 14 times less TLL gene transcripts (data not shown).Expression levels of mRNA transcripts of various housekeeping genes weresimilar in both healing gal-3^(+/+) and gal-3^(−/−) as detected by bothmicroarray technology (FIG. 12), and semi-quantitative RT-PCR (FIG. 12,GAPDH is D-glyceraldehyde-3-phosphate dehydrogenase; RPS29 is ribosomalprotein S29; ODC is ornithine decarboxylase).

To determine whether the expression level of the galectin-7 protein isalso reduced in healing corneas of gal-3^(−/−) mice, western blotanalysis using detergent extracts of healing gal-3^(+/+) and gal-3^(−/−)corneas (FIG. 13A) and immunohistochemical studies with ananti-galectin-7 polyclonal antibody using paraffin sections derived fromcorneas of gal-3^(+/+) and gal-3^(−/−) mice (FIG. 13B) were performed.The immunoreactivity was graded as intense (+++), moderate (++), weak(+) or negative (−). Significantly less galectin-7 immunoreactivity wasdetected in migrating epithelia of healing gal-3^(−/−) corneas comparedto those of healing gal-3^(+/+) corneas: gal-3^(+/+):+++36/42, ++5/42; +or less 1/42; gal-3^(−/−):+++3/42., ++26/42, + or less 13/42. Also,gal-3^(−/−) mouse embryonic fibroblasts (MEF) grown in cell cultureexpressed reduced levels of galectin-7 compared to gal-3^(+/+) MEFcultures (FIG. 13C).

Example 4 Exogenous Galectin-3 Stimulates the Re-Epithelialization ofCorneal Wounds in Wild Type and Galectin-3 Deficient Mice

Having demonstrated that corneal epithelial wound closure rate isperturbed in gal-3^(−/−) mice (Example 2), it was of interest todetermine whether exogenous galectin-3 would stimulate there-epithelialization of healing corneas in organ culture. In this study,corneas of gal-3^(+/+) and gal-3^(−/−) mice with alkali-burn wounds wereincubated in serum free media in the presence and absence of varyingamounts of recombinant galectin-3.

Recombinant full-length human galectin-3 was produced in Eschericia coliand purified as described previously (Yang et al., Biochemistry 37:4086,1998). Alkali-burn wounds (2 mm diameter) were produced on both eyes ofanesthetized mice using alkali-soaked filter discs as described inExample 2. Following injury, the animals were sacrificed and the eyeswere excised and incubated in the presence or absence of exogenousgalectin-3 for 18 to 20 hours. The left eyes of animals served ascontrols and were incubated in serum free media alone. The right eyeswere incubated in serum free media containing various test reagentsincluding: (i) galectin-3 (5 to 20 μg/ml), (ii) galectin-3 (10 μg/ml)plus 0.1 M β-lactose, (iii) galectin-3 (10 μg/ml) plus 0.1 M sucrose,(iv) 0.1 M β-lactose, or (v) 0.1 M sucrose. At the end of the healingperiod, the remaining wound areas were stained, photographed andquantified as described in Example 2 using SIGMASCAN™ software (SPSSScience of Chicago, Ill.). Each group contained a minimum of three eyesand all experiments were performed at least twice.

The exogenous galectin-3 had no influence on the rate ofre-epithelialization of corneal wounds in gal-3^(−/−) mice (FIG. 14A),but it stimulated the rate of wound closure in a concentration-dependentmanner in gal-3^(+/+) mice (FIG. 14B) at 10 μg/ml and 20 μg/mlconcentration (0 and 5 μg/ml: 0.090±0.010 mm²/h; 10 μg/ml: 0.129±0.010mm²/h; 20 μg/ml: 0.154±0.004 mm²/h; mean±SEM of at least twoexperiments, N=7 or more). As shown in FIG. 15, the stimulatory effectof galectin-3 on corneal epithelial wound closure in gal-3^(+/+) micewas specifically inhibited by β-lactose but not sucrose (10 μg/mlgalectin-3: 0.127±0.010 mm²/h; 10 μg/ml galectin-3 plus 0.1 M β-lactose:0.103±0.014 mm²/h; 10 μg/ml galectin-3 plus 0.1 M sucrose: 0.130±0.003mm²/h. All values represent mean±SEM of at least two experiments, N=7 ormore).

Example 5 Exogenous Galectin-7 Stimulates the Re-Epithelialization ofCorneal Wounds in Wild Type and Galectin-3 Deficient Mice

In a separate study, comparison of the gene expression patterns ofnormal and healing corneas of gal-3^(+/+) mice using cDNA microarrays(i.e., as in Example 3) revealed that in healing corneas, expression ofgalectin-7 is markedly up-regulated. These findings in conjunction withthe studies described in Example 3 showing that galectin-7 expression isdown-regulated in the healing cornea of gal-3^(−/−) mice, led to thedesign of experiments to determine whether exogenous galectin-7 wouldstimulate the re-epithelialization of healing corneas in organ culture.In this study, corneas of gal-3^(−/−) mice with alkali-burn wounds wereincubated in serum free media in the presence and absence of varyingamounts of recombinant galectin-7.

Recombinant full-length human galectin-7 was produced in Eschericia coliby cloning the cDNA (available as an EST clone from American TypeCulture Collection of Manassas, Va.) into the pET25b plasmid (availablefrom Novagen, Madison, Wis.). Alkali-burn wounds (2 mm diameter) wereproduced on both eyes of anesthetized animals using alkali-soaked filterdiscs as described in Example 2. Following injury, the animals weresacrificed and the eyes were excised and incubated in the presence orabsence of exogenous galectin-7 for 18 to 20 hours. The left eyes ofanimals served as controls and were incubated in serum free media alone.The right eyes were incubated in serum free media containing varioustest reagents including: (i) galectin-7 (20 μg/ml), (ii) galectin-7 (20μg/ml) plus 0.1 M β-lactose, or (iii) galectin-7 (20 μg/ml) plus 0.1 Msucrose. At the end of the healing period, the remaining wound areaswere stained, photographed and quantified as described in Example 2using SIGMASCAN™ software (SPSS Science of Chicago, Ill.). Each groupcontained a minimum of six eyes and all experiments were performed atleast twice.

As shown in FIG. 16, exogenous galectin-7 stimulated the rate of woundclosure (media alone: 0.036±0.006 mm²/h; 20 μg/ml galectin-7:0.072±0.004 mm²/h; mean±SEM of at least two experiments, N=10 or more).As shown in FIG. 16, the stimulatory effect of galectin-7 on cornealepithelial wound closure was specifically inhibited by β-lactose but notby sucrose (20 μg/ml galectin-7: 0.072±0.004 mm²/h; 20 μg/ml galectin-7plus 0.1 M β-lactose: 0.050±0.004 mm²/h; 20 μg/ml galectin-7 plus 0.1 Msucrose: 0.079±0.007 mm²/h. All values represent mean±SEM of at leasttwo experiments, N=9 or more). As shown in FIG. 16, the rate of woundclosure was further enhanced (0.094±0.003 gal-3^(+/+) mm²/h) whenexogenous galectin-7 was added to the injured corneas of gal-3^(+/+)mice instead of gal-3^(−/−) mice.

Example 6 Skin Epithelial Wound Closure in Wild Type and Galectin-3Deficient Mice

Gal-3^(+/+) and gal-3^(−/−) mice are anesthetized by an intraperitonealinjection of 1.25% Avertin (0.2 ml/10 g body weight). Prior to lasertreatment, hair is shaved off from the dorsal region using a razorblade. Six millimeter transepithelial dorsal skin wounds are made usingthe excimer laser (Summit Technology of Waltham, Mass.). After surgery,antibiotic ointment is applied to the wound surface and buprenorphine (2mg/kg body weight) is given subcutaneously to minimize post-surgicalpain. The wounds are allowed to partially heal in vivo, and are examined24, 48, and 72 hours after surgery. At the end of the healing period,the mice are again anesthetized by an intraperitoneal injection of 1.25%Avertin (0.2 ml/10 g body weight), wound areas are photographed and thenquantitated using a Sigma scan software. The wound closure rates betweenthe two groups of animals (i.e., gal-3^(+/+) and gal-3^(−/−) mice) arecompared. The animals are then sacrificed by carbon dioxide inhalationor an overdose of pentobarbital.

Example 7 Effect of Exogenous Galectin-3 on the Re-Epithelialization ofSkin Wounds

Animals (Mice: 57BL/6 and 129 mixed genetic background; Age: six toeight weeks old; Gender: mixed) are anesthetized by an intraperitonealinjection of 1.25% Avertin (0.2 ml/10 g body weight). Prior to lasertreatment, hair is shaved off from the dorsal region using a razorblade. Two 6-mm transepithelial dorsal skin wounds (one on each side)are made using the excimer laser (Summit Technology of Waltham, Mass.).After surgery, antibiotic ointment is applied to the wound surfaces andbuprenorphine (2 mg/kg body weight) is given subcutaneously to minimizepost-surgical pain. The wounds are then allowed to partially heal invivo. Every 4-6 hours, an ointment containing galectin-3 is applied tothe right wound and carrier only is applied to the left wound whichserves as a control. At the end of the healing period (24 to 48 hours),the animals are anesthetized by an intraperitoneal injection of 1.25%Avertin (0.2 ml/10 g body weight), wound areas are photographed andquantitated using a Sigma scan software. The wound closure rates betweenthe two groups of animals (galectin-3 treated and control) are compared.The animals are then sacrificed by carbon dioxide inhalation or anoverdose of pentobarbital.

Example 8 Effect of Exogenous Galectin-7 on the Re-Epithelialization ofSkin Wounds

Animals (Mice: 57BL/6 and 129 mixed genetic background; Age: six toeight weeks old; Gender: mixed) are anesthetized by an intraperitonealinjection of 1.25% Avertin (0.2 ml/10 g body weight). Prior to lasertreatment, hair is shaved off from the dorsal region using a razorblade. Two 6-mm transepithelial dorsal skin wounds (one on each side)are made using the excimer laser (Summit Technology of Waltham, Mass.).After surgery, antibiotic ointment is applied to the wound surfaces andbuprenorphine (2 mg/kg body weight) is given subcutaneously to minimizepost-surgical pain. The wounds are then allowed to partially heal invivo. Every 4-6 hours, an ointment containing galectin-7 is applied tothe right wound and carrier only is applied to the left wound whichserves as a control. At the end of the healing period (24 to 48 hours),the animals are anesthetized by an intraperitoneal injection of 1.25%Avertin (0.2 ml/10 g body weight), wound areas are photographed andquantitated using a Sigma scan software. The wound closure rates betweenthe two groups of animals (galectin-7 treated and control) are compared.The animals are then sacrificed by carbon dioxide inhalation or anoverdose of pentobarbital.

CONCLUSION

It has been demonstrated that galectin-3 and galectin-7 play a role inthe re-epithelialization of corneal wounds. In Example 1immunohistochemical studies revealed that following injury, galectin-3is located in high density at sites of corneal epithelial cell-matrixadhesion, an ideal location for influencing cell-matrix interactions andhence cell migration. In Example 2, the re-epithelialization of cornealwounds was shown to be significantly slower in the galectin-3 deficientmice compared to that in wild-type mice. In Example 3, it was shown thatfollowing injury, expression levels of galectin-7 are significantlyreduced in galectin-3 deficient mice compared to wild-type mice. InExamples 4 and 5, exogenous recombinant galectin-3 and galectin-7 wereshown to stimulate the re-epithelialization of corneal wounds ingal3^(+/+) mice. It was further demonstrated in Example 1 that thestimulatory effect of galectin-3 on the rate of corneal epithelial woundclosure can be almost completely-abrogated by a competing disaccharide(β-lactose), but not by an irrelevant disaccharide (sucrose). This finalresult suggests that the carbohydrate recognition domain (CRD) isdirectly involved in the beneficial effect of the exogenous galectin-3on wound closure.

Without wishing to be bound to any particular theory regarding themechanism by which galectin-3 and galectin-7 may influencere-epithelialization of corneal wounds, the following suggestions arepresented.

As mentioned earlier, galectin-3 is thought to mediate cell-cell andcell-matrix interactions by binding to complementary glycoconjugatescontaining polylactosamine chains found in many ECM and cell surfacemolecules such as certain isoforms of fibronectin, laminin, andintegrins (Liu, Clin. Immunol. 97:79, 2000 and Perillo, supra). However,the finding presented herein that exogenous galectin-3 does notaccelerate the re-epithelialization of wounds in gal3^(−/−) mice (seeExample 4) suggests that intracellular galectin-3 contributessignificantly to the process of wound healing, most probably, byinfluencing the expression of specific cell surface and/or ECMreceptors, which in turn influence cell-matrix interactions and cellmigration. This idea is consistent with published studies in whichgalectin-3 was stably overexpressed in breast carcinoma cell lines,resulting in elevated levels of α4β7 and α6β1 integrins and enhancedadhesion to various ECM molecules including laminin, fibronectin, andvitronectin as compared with parental cell lines expressing little or nogalectin-3 (Warfield, supra and Mattarese, supra). In another study(Dudas et al., Gastroenterology 118:1553, 2000), colon cancer carcinomacell lines transfected with galectin-3 expressed elevated levels of aspecific mucin, MUC2, a major ligand of the lectin itself (Bresalier etal., Cancer Research 56:4354, 1996). The fact that the stimulatoryeffect of exogenous galectin-3 on the rate of re-epithelialization ofwounds in gal3^(+/+) mice is lactose inhibitable raises an intriguingpossibility that intracellular galectin-3 may in fact regulateglycosylation of the proteins which serve as cell surface or ECMreceptors of the lectin itself. That intracellular galectin-3 has thepotential to act on the nuclear matrix to influence complex biologicalprocesses is also suggested by findings that under certain conditionsthe lectin can be found associated in the nucleus with ribonucleoproteincomplexes and can act as a pre-mRNA splicing factor (Dagher et al.,Proc. Natl. Acad. Sci. USA 92:1213, 1995). Also, Wang et al. havedemonstrated that in prostate adenocarcinoma cells, galectin-3 isassociated with the nuclear matrix and binds with both single-strandedDNA and RNA (Wang et al., Biochem. Biophys. Res. Commun. 217:292, 1995).

Analysis of gene expression patterns of corneas of healing gal3^(+/+)and gal3^(−/−) mice using mouse cDNA microarrays revealed that healingcorneas of gal3^(−/−) mice expressed markedly reduced levels ofgalectin-7 compared to those of wild-type mice (see Examples 3 and 5).Galectin-7 was first reported in 1994 (Barondes, supra) and is not aswell characterized as galectin-3. Unlike galectin-3, galectin-7 exhibitsa remarkable degree of tissue specificity. In adult animals, itsexpression is restricted to epithelia that are or are destined to becomestratified (Timmons et al., supra). The protein is thought to beinvolved in cell-matrix and cell-cell interactions and in apoptosis(Leonidas, Biochemistry 37:13930, 1998 and Bernerd et al., Proc. Natl.Acad Sci. USA 96:11329, 1999). In general, an inverse correlation existsbetween galectin-7 expression and keratinocyte proliferation, andgalectin-7 expression is abrogated in SV40 transformed keratinocytes aswell as in cell lines derived from epidermal tumors. The discoverydescribed herein that exogenous galectin-3 does not stimulatere-epithelialization of wounds in gal3^(−/−) corneas and that healinggal3^(−/−) corneas contain reduced levels of galectin-7 suggests thatgalectin-3 may influence the re-epithelialization of wounds, at least inpart, by modulating galectin-7. Indeed, it has been found that unlikegalectin-3, galectin-7 accelerated re-epithelialization of wounds ingal3^(−/−) corneas in a lactose-inhibitable manner. Also, mouseembryonic fibroblasts of gal3^(−/−) mice expressed reduced level ofgalectin-7.

Regardless of the mechanisms involved, the findings that both galectin-3and galectin-7 stimulate re-epithelialization of corneal wounds havebroad implications for the treatment of epithelial wounds andnon-healing epithelial wounds in particular. At present, treatment ofpersistent epithelial defects of the cornea is a major clinical problem.Moreover, the need continues for effective treatment ofpost-transplantation wounds, chronic wounds in the elderly, decubitusulcers, and venous stasis ulcers of the skin. A number of growth factors(e.g., EGF, TGF, FGF, KGF, HGF) known to stimulate cell proliferation,have been tested for usefulness in corneal as well as cutaneousepithelial wound healing with overall disappointing results (Eaglstein,Surg. Clin. North Am. 77:689, 1997; Singer and Clark, N. Engl. J. Med.341:738, 1999; Zieske and Gipson, pp. 364-372 in “Principle and Practiceof Opthalmology” Ed. by D. M. Albert and F. A. Jakobiec, W.B. SaundersCompany, Philadelphia, Pa., 2000; Schultz et al., Eye 8:184, 1994;Kandarakis et al., Am. J. Opthalmol. 98:411, 1984; and Singh and Foster,Am. J. Opthalmol. 103:802, 1987). The extent of acceleration ofre-epithelialization of wounds was far less in most of these studiesusing growth factors than that observed with galectins in the currentstudy. Also, the epithelium of corneas treated with growth factors suchas EGF is hyperplastic (Singh and Foster, Cornea 8:45, 1989), a clearlyundesirable condition. In this respect, the clinical potential ofgalectin-3 and galectin-7 may be more attractive than that of growthfactors because the lectins have not been shown to induce cell mitosisin epithelial cells. Over the last decade, the potential of excimerlaser keratectomy to modify the corneal profile for correction of myopiahas been realized. Thousands of such procedures are performed each weekproviding myopic individuals freedom from eye glasses and contactlenses. In view of the fact that 25-30% of the adult populationworldwide is myopic, it has been estimated that nearly half a millionsuch procedures will be performed in the U.S. alone in a given year. Insome cases, following excimer laser surgery, there is a delay inepithelial healing. Such a delay is highly undesirable because it putsthe cornea at risk of developing postoperative haze, infectiouskeratitis and ulceration. Again, galectin-based treatments may helppromote re-epithelialization of wounds in such cases.

OTHER EMBODIMENTS

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of the specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope of theinvention being indicated by the following claims.

1. (canceled)
 2. A method for the therapeutic treatment of epithelialwounds in mammals comprising administering to a mammal afflicted with anepithelial wound a therapeutically effective amount of a galectin-7protein.
 3. The method according to claim 2, wherein said epithelialwound is a persistent epithelial defect or a recurrent epithelialerosion.
 4. The method according to claim 2, wherein said epithelialwound is an epithelial lesion or an epithelial erosion.
 5. The methodaccording to claim 2, wherein said epithelial wound is selected from thegroup consisting of ulcers, blisters, burns, sores, boils, cuts,abrasions, and lacerations.
 6. The method according to claim 2, whereinsaid epithelial wound is a corneal wound.
 7. The method according toclaim 6, wherein said corneal wound is a persistent corneal defect or arecent corneal erosion.
 8. The method according to claim 6, wherein saidcorneal wound was caused by excimer laser keratectomy.
 9. The methodaccording to claim 2, wherein said epithelial wound is a skin wound or awound to the gastrointestinal tract. 10-14. (canceled)
 15. The method ofclaim 2, wherein the galectin-7 protein includes the amino acid sequenceof SEQ ID NO:2.
 16. The method of claim 2, wherein the galectin-7protein includes an amino acid sequence that is substantially identicalto the amino acid sequence of SEQ ID NO:2.
 17. The method of claim 2,wherein the galectin-7 protein includes a galectin-7 galactoside-bindingdomain. 18-35. (canceled)
 36. A method for the therapeutic treatment ofepithelial wounds in mammals comprising administering to a mammalafflicted with an epithelial wound a therapeutically effective amount ofa substance that influences the expression of a galectin-7 protein.37-38. (canceled)